5′-AZA-2′-Deoxycytidine Induces p21/WAF Expression by Demethylation of p73 Leading to p53-Independent Apoptosis in Myeloid Leukemia.

The DNA methylation inhibitor 5′-Aza-2-deoxycytidine (5-Aza-CdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute and chronic myeloid leukemia (AML and CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition effect. We found that 5-Aza-CdR induces apoptosis in AML cells (both, p53 mutant and wildtype) but not in epithelial or normal peripheral blood mononuclear cells (PBMC). Cell death was accompanied by activation of the mitochondrial apoptosis pathway as shown by early release of cytochrome c and AIF and loss of the mitochondrial membrane potential Δψm. Activation of caspase-3 but not -6 and -8 was detectable using western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21 which correlated with the arrest of AML cells in the G1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but was mediated by 5-Aza-CdR-induced reexpression of the tumor suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells which were resistent towards 5-Aza-CdR. Therefore, 5-Aza-CdR-mediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells.